PROJECT SUMMARY/ABSTRACT Vascular hyperpermeability is a hallmark of inflammation Current therapy interferes with mechanisms involved in onset of hyperpermeability. We and other investigators are focusing on mechanisms that terminate hyperpermeability. Because many negative effects of hyper- permeability are due to its persistence beyond what is required for preserving organ function, we propose to elucidate mechanisms that terminate hyperpermeability and thereby restore microvascular barrier properties, while retaining its beneficial effects. Based on a) protein traffic mechanisms, b) our demonstration that eNOS translocation to cytosol is necessary for onset of hyperpermeability, c) the demonstration that VASP (vasodilator stimulated phosphoprotein) is fundamental for cell adhesion and endothelial barrier properties, and d) our preliminary data that selective stimulation of Epac (exchange protein activated by cAMP) returns eNOS to the cell membrane, we will test the central hypothesis that the signaling that leads to endothelial hyperpermeability initiates a delayed increase in [cAMP], which causes VASP-mediated translocation of eNOS and Epac to the cell membrane leading to inactivation of hyperpermeability. We will test this hypothesis through 4 Specific Aims; in each Aim measurement of permeability will be an end-point in vivo or in vitro: SA1. To determine whether during hyperpermeability the activity of cytoplasmic and not plasma membrane eNOS causes the increase in cAMP required for inactivation of hyperpermeability. We will assess inactivation of hyperpermeability and measure [cAMP] as a function of [NO] in cells with eNOS expressed either in the cytosol or in the cell membrane, and in primary endothelial cells (EC). SA2. To determine whether a G-protein-coupled receptor (GPCR) mediates the increase in [cAMP]. This aim will study a plausible alternative mechanism to cytosolic [NO] as the cause for the increase in [cAMP]. SA3. To determine the role of VASP phosphorylation in contributing to eNOS and Epac translocation to cell membrane. In wild type and in VASP-KO endothelial cells and mice, we will test the cause-effect relation between VASP phosphorylation and inactivation of hyperpermeability. We will stimulate eNOS translocation with 8-cPT-2-O-Me-cAMP, a selective activator of Epac. SA4. To determine the signaling interactions between VASP phosphorylation and VASP S-nitrosation in the regulation of hyperpermeability. S-nitrosation is increasingly recognized as an important posttranslational regulatory modification in EC. Whether S- nitrosation promotes inactivation is unknown. The results of this conceptually innovative proposal will influence current paradigms in microvascular permeability and will provide the basis for developing new therapies for treating vascular inflammation.